The overall objectives of this program focus on the genesis and assembly of the red cell, with emphasis on the membrane skeleton. A continuing long term objective is to understand the pathophysiologic mechanisms of hemolytic anemia. New emphasis is placed on exploring novel role(s) of prototypical red cell skeletal proteins in intracellular structures in erythroid progenitors and in nonerythroid cells. New perspectives on skeletal assembly and function include model studies of kidney epithelial cells, and of the budding yeast, S.cerevisiae. To achieve these broad goals, six complementary approaches are proposed: 1. Characterize the structure and function of a complex repertoire of developmentally-regulated skeletal protein 4.1 isoforms, by molecular studies of the gene and its multiple alternative transcripts, and via use of transgenic mouse technology; 2. Explore the role of skeletal proteins in plasma membrane remodeling and in nuclear and centrosomal function during erythropoiesis, and study the role of interactions between erythroid progenitors and marrow microenvironment in regulating erythroid differentiation; 3. Characterize a novel spectrin-based skeletal protein complex of the Golgi, and investigate its dynamics of assembly and its function in model kidney epithelial cells; 4. Explore general principles of skeletal assembly and function by cloning conserved homologs from yeast and mammals, and investigating function by uniquely combining powerful yeast genetics with biophysical techniques developed in this program for analysis of red cell membranes; 5. Analyze pathophysiologic mechanisms of red cell loss in thalassemia, using murine models developed in this program to explore the hypothesis that a-globin chain accumulation, by provoking oxidant damage at the membrane, activates an apoptotic program ultimately manifested as ineffective erythropoiesis; 6. Examine functional consequences of specific interactions between red cell membrane proteins and proteins elaborated by intraerythrocytic stages of the malarial parasite, Plasmodium falciparum, especially as they affect red cell deformability and cytoadherence. Finally, supporting the projects is a mouse core which will prepare transgenic and knockout mice, as well as maintain mouse lines. Application of this broad range of expertise in molecular biology, genetics, biochemistry, cell biology, and biophysics should provide a better understanding of fundamental principles of membrane organization in eukaryotic cells, and may eventually provide in sights into management of hemolytic anemias in which membrane structure, function, and development are deranged.